Trypanosoma Cruzi Affects The Sensory Biology of Triatoma Dimidiata (Hemiptera: Reduviidae)

Irving J. May-Concha (  irving.may@correo.uady.mx ) Regional Centre of Research Dr Hideyo Noguchi: Centro de Investigaciones Regionales Dr Hideyo Noguchi Maryrose J. Escalante Talavera Regional Centre of Research Dr Hideyo Noguchi: Centro de Investigaciones Regionales Dr Hideyo Noguchi Jean-Pierre Dujardin Institut de recherche pour le développement: Institut de recherche pour le developpement Etienne Waleckx Regional Centre of Research Dr Hideyo Noguchi: Centro de Investigaciones Regionales Dr Hideyo Noguchi


Insects
Laboratory-reared T. dimidiata came from a colony maintained for the past 10 years at the Parasitology Laboratory of the Regional Research Center Dr. Hideyo Noguchi, Autonomous University of Yucatan. New insects have been periodically added to this colony to avoid inbreeding depression. The colony is reared and maintained at 27 ± 1°C, 70 ± 5% RH, a photoperiod of 12: 12 (L: D) h, and insects are fed on immobilized pigeons (Columba livia). The domestic and sylvatic populations were composed of insects collected during entomological surveillance inside and outside human dwellings of the rural village of Teya (21.05º N, 89.07º W), Yucatan, Mexico, and in the sylvatic habitat surrounding this village, respectively [73][74]. The study was approved by the Institutional Bioethics Committee of the Autonomous University of Yucatan.

Trypanosoma cruzi
For infection of triatomines, the "V strain", a TcI strain of T. cruzi maintained in the laboratory by cyclical passages in BALB/c adult mice was used.

Infection of the laboratory-reared triatomines
After a two-week starvation period, the initial infection of the laboratory-reared triatomines was carried out with nymphs that had just molted to their 5th instar. Nymphs were fed ad libitum on BALB/c mice 15 days after these were infected with 1×10 6 parasites ml − 1 of blood (i.e., during the parasite's exponential stage of growth; [63]). Approximately 30 days after infection, we corroborated infection status through examination of a fecal drop observed under a light microscope at 40× magni cation. Control group insects were fed under the same conditions on non-infected mice. The nymphs of both groups were maintained under rearing conditions and were fed fortnightly on infected/non-infected mice until they molt to the adult stage. For T. dimidiata collected in natural conditions (i.e. domestic and sylvatic populations), T. cruzi infection status was assessed by amplifying parasite DNA from each bug midgut by PCR using TCZ primers, as previously described [75].

Antennal preparation
We examined a total of 130 antennae of T. cruzi-infected and non-infected females and males from the sylvatic, domestic and laboratory-reared populations of T. dimidiata (Table 1). One antenna per specimen was removed using ne forceps and scissors. Antennae were processed with sodium hydroxide 4% for 6 hr at 60°C and then neutralized with glacial acetic acid 5% for 2 min. This procedure allowed cuticle diaphanization and allowed the identi cation and counting of the sensilla using a stereo microscope Zeiss Primostar® at 400×. The number and type of sensilla on antennal segments was counted manually using a procedure reported previously [33]. The ventral side of the three distal segments of the antennae (P: pedicel, F1: agellum 1, and F2: agellum 2) was evaluated, by identifying and counting the following sensilla: bristles (BR), thin-walled trichoid (TH), thick-walled trichoid (TK), and basiconic (BA) (nomenclature according to Catalá and Scho el [36]), thus giving a total of 12 morphological variables.

Effect of the infection with T. cruzi on the AP of T. dimidiata
Differences of each sensillum on the three antennal segments between infected and non-infected insects' overall populations, within each sex, within each population, and within each sex within each population, are summarized in Table 2. Table 2 Comparisons of the abundances of each sensillum between infected and non-infected insects overall populations, within each sex, within each population, and within each sex within each population of Triatoma dimidiata. BR: bristles; BA: basiconic; TH: thin-walled trichoid; TK: thick-walled trichoid. F: female and M: male. I: infected; NI: non-infected. D: domestic, S: Sylvatic, and L: Laboratory reared. Asterisks represent a signi cant difference between infected and non-infected insects (P < 0.05*; P < 0.01**; P < 0.001***; --no difference).
Whitin females (I females vs NI females)  3. 3.2.3. Within each population. In the domestic population, when infected and non-infected insects were compared, a signi cant increase in the number of BR sensilla on pedicel (P-BR) was observed in infected insects (Kruskal-Wallis test, P = 0.01). On the other side, signi cant decreases in the number of TH and TK sensilla on pedicel; BR, BA, TK sensilla on agellum 1, and BR, BA, TH sensilla on agellum 2 were observed in infected insects compared to non-infected insects (Kruskal-Wallis test, P < 0.05 in all cases). Additionally, the two-way PERMANOVA test revealed that the infection with T. cruzi affected the AP of the domestic population (F = 7.15; P = 0.0001), while the sex and the interaction infection*sex did not have signi cant effects (F = 1.51; P = 0.177 and F = 1.188; P = 0.299, respectively; Table 3A).
In the sylvatic population, when infected and non-infected insects were compared, signi cant increases in the number of BA sensilla on pedicel; BR, BA, and TK sensilla on agellum 1; BR, BA, TH and TK sensilla on agellum 2 were observed in infected insects (Kruskal-Wallis test, P < 0.05 in all cases). The two-way PERMANOVA test revealed that the infection with T. cruzi and the sex affected the AP of the sylvatic population (F = 7.41; P = 0.0001 and F = 4.28; P = 0.002, respectively), while the interaction infection*sex did not have signi cant effect (F = 0.368; P = 0.125; Table 3B).
Finally, in the laboratory-reared population, when infected and non-infected insects were compared, no difference in the number of sensilla were observed (Kruskal-Wallis test, P > 0.05 in all cases). In the same way, the two-way PERMANOVA test did not reveal signi cant effects of the infection with T. cruzi, of the sex and of the interaction infection*sex on the AP of laboratory-reared insects (P > 0.05; Table 3C).

3.2.4.
Within each sex within each population. Differences in the abundances of each sensillum on the three antennal segments between infected and non-infected insects within each sex within each population are shown in Table A1 (Supplementary data) and are summarized in Table 2.
Domestic population. The antennae of infected females of the domestic population showed a signi cant decrease in the number of TH sensilla on pedicel and agellum 2, and in the BA sensilla on agellum 2, compared to non-infected females (Kruskal-Wallis, P < 0.05 in all cases). On the other side, infected males of the domestic population showed an increase in the number of BR sensilla on pedicel (P-BR), compared to non-infected males (Kruskal-Wallis test, P = 0.01). Moreover, infected males of the domestic population showed a decrease in the BR sensilla on agellum 1 and agellum 2, and in the BA sensilla on agellum 2, compared to non-infected males (Kruskal-Wallis, P < 0.05 in all cases).
Sylvatic population. The antennae of infected females of the sylvatic population showed a signi cant increase in the number of BA sensilla on the three segments of the antennae, in the BR sensilla on agellum 1 and agellum 2, and in the TK sensilla on agellum 2, compared to non-infected females (Kruskal-Wallis test, P < 0.05 in all cases). On the other side, infected males of the sylvatic population showed an increase in the BA sensilla on pedicel, in the BR and TK sensilla on agellum 1, and in the BR and TH sensilla on agellum 2, compared to non-infected males (Kruskal-Wallis test, P < 0.05 in all cases).
Laboratory-reared population. In the laboratory-reared population, there were no differences in the number of sensilla between infected and non-infected females and males (Kruskal-Wallis test, P > 0.05).

Effect of the infection with T. cruzi on the sexual dimorphism of T. dimidiata.
Differences in the abundances of each sensillum between non-infected females and males, and between infected females and males overall populations, and within each population, are summarized in Table 4. Table 4 Comparisons of the abundances of each sensillum between infected females and males and between non-infected females and males overall populations, and within each population of Triatoma dimidiata. BR: bristles; BA: basiconic; TH: thin-walled trichoid; TK: thick-walled trichoid. F: female and M: male. I: infected; NI: non-infected. D: domestic, S: Sylvatic, and L: Laboratory reared. Asterisks represent a signi cant difference between infected and non-infected insects (P < 0.05*; P < 0.01**; P < 0.001***; --no difference).   [93], as has been shown in the insect Cimex lectularius L. [94]. In our study, variations in the olfactory sensitivity because of T. cruzi infection in the domestic and sylvatic populations is suggested. Indeed, in these populations, infected and non-infected insects showed signi cant differences in the number of some speci c chemoreceptors. Surprisingly, infected insects of the domestic populations showed a decrease in some chemoreceptors. Conversely, in the sylvatic population, the infection with T. cruzi increased in all cases the number of chemoreceptors. If these differences between populations are not easy to explain, in general, the infection with T. cruzi signi cantly increased the number of chemoreceptors, which may be linked to an improved capacity for dispersal and invasion of different habitats [79,90], and better e ciency in the perception of odor molecules in the search of distant mates and hosts and for ight dispersal in search of new habitats, as it has been suggested by other authors [80, [95][96][97][98], thus conferring an advantage to T. cruzi.
Although several studies show the effect of T. cruzi on behavioral changes [53, 61-65], so far, no study has analyzed whether there are differences in the olfactory physiology between infected and non-infected and a possible correlation of behavioral changes with their AP. In this context, it is possible to ask if the behavioral changes may be due to the AP modi cation in infected insects. Although the present work did not aim to answer this question, considering the recently reviewed information and the results of this study, we could suggest a possible correlation of behavioral changes with the AP as it has been suggested by May-Concha et al. [34]. Therefore, it could be interesting to evaluate the olfactory system of infected insects and non-infected towards chemical cues to nd components of attractive blends that could contribute to the list of volatile compounds that modulate the behavior between infected and noninfected insects, to design better strategies for behavioral manipulation of this triatomines. May-Concha [102] provides information on a chemical signal produced during T. dimidiata mating since fewer mating attempts were observed when the opening of female glands was occluded. Besides, that study describes a chemical signal which promotes the attraction of males to volatiles emitted by females and to mating couples [30]. On the other side, based on previous works on olfactory receptors [24][25][26], we propose that the increased number of TH chemo-sensilla in infected males could suggest a greater e ciency in the perception of odor molecules involved in sexual communication compared with infected females. In contrast, the increased number of BA chemo-sensilla in infected females could suggest a greater e ciency in the perception of host odors compared with infected males. Therefore, the increase in the odor perception in infected insects probably elicits a positive effect on population dynamics and could affect the vectorial transmission of T. cruzi. However, to achieve depth in the knowledge on this subject, studies on the effects of the infection with T. cruzi on the behavior of aggregation, alarm, sexual pair, feeding, excretion/defecation, and host foraging in T. dimidiata should be carried out.

Availability of data and materials
The datasets used and/or analyzed during the present study available from the corresponding author upon reasonable request.
Ethics approval and consent to participate Not applicable.